Biologically active materials, such as proteins, are important catalysts for industrial and analytical chemistry because they are highly specific to particular reagents and because they exhibit high catalytic activity and speed of reaction. Enzymes, antibodies, and enzyme-antibody conjugates are sub-classes of proteins. However, biologically active proteins are often difficult to isolate and expensive to make and use. It is usually desirable to utilize proteins in an immobilized form in which biologically active proteins are attached to a solid carrier which confines the active proteins and permits their reuse. Several means of immobilizing biologically active proteins on solid carriers are known, such as adsorption, ionic bonding, entrapment, and covalent bonding.
Proteins, of which enzymes and antibodies are sub-classes, are relatively large molecules that contain positively charged, negatively charged, and uncharged non-ionic portions. Ionicly bound proteins are produced when a charged portion of the protein comes in close contact to an oppositely charged portion of a solid carrier surface. Under many conditions this attachment is irreversible. Adsorptively bound proteins are produced when the uncharged portion of the protein comes in close contact to an uncharged, non-ionic portion of a solid carrier surface. Several ionic and adsorptive attachments may be necessary to irreversibly bind a single protein.
Covalent bonds that immobilize proteins are formed by linking amino or carboxyl groups, which are present in every enzyme, with polar functional groups attached to the carrier. The functional groups can be derived from components normally present in the material that forms a carrier substrate or the functional groups can be added to the carrier. Suitable functional groups include carboxyl groups, amino groups, sulphonic acid groups, imino groups, thio groups, hydroxyl groups, azo groups, epoxy groups, aldehyde groups, acid chloride groups, activated carbonyl groups, pyridyl groups, and phosphoryl groups. The functional groups may be further activated by chemical treatment to enhance their ability to join with the amino or carboxyl groups present in the protein molecules.
Known techniques for covalently immobilizing proteins on porous carriers have significant limitations. For example, large amounts of biologically active material can be immobilized inside a bulk carrier, such as a block of porous material or thick porous membrane, but the depth and physical properties of the protein layers so immobilized are difficult to control. Also, diffusion into and out of blocks and relatively thick membranes is slow.
By another technique, biologically active material, such as enzymes, antibodies, or enzyme-antibody conjugates can be immobilized in small amounts as a thin layer inside thin membranes or as a thin layer on the surface carrier membranes, but the limited amount of biologically active material which can be deposited into the thin layers is often insufficient to carry out a desired chemical reaction. Further, biological materials tend to deactivate over time and a carrier with only a small amount of active material may not function over a period of time long enough to be practical.